The insulin-like growth factors, IGF-I and IGF-II, are proteins that are homologous to insulin which stimulate cell survival and proliferation by binding to and activating IGF-I receptors. The IGFs also bind with high affinity to six secreted IGF-binding proteins (IGFBPs). The IGFBPs regulate IGF biological activity by forming complexes that prevent them from activating IGF-I receptors. IGFBP-3 also can inhibit cell proliferation and induce apoptotic cell death independently, without binding IGFs. During the past year, our ongoing studies of the regulation and biological role of the IGFBPs have focused on understanding the IGF-independent mechanisms by which IGFBP-3 induces apoptosis in human prostate cancer cells.[unreadable] [unreadable] We previously reported that an IGFBP-3 mutant (6m-IGFBP-3) that does not bind IGF-I or IGF-II can induce apoptosis in PC-3 human prostate cancer cells as effectively as wild-type IGFBP-3. The IGF-independent mechanisms by which IGFBP-3 acts are poorly understood. Secreted IGFBP-3 presumably interacts with cell surface receptors to activate signal transduction pathways or to be internalized. Internalized IGFBP-3 can translocate to the nucleus and interacts with the nuclear retinoid X receptor RXR. We initiated studies to determine whether nuclear localization is essential for IGFBP-3 to induce apoptosis. We fused IGFBP-3 to yellow fluorescent protein (YFP), and mutated the nuclear localization signal (NLS) of IGFBP-3 to the corresponding sequence in IGFBP-1 (MDGEA) since IGFBP-1 does not localize to the nucleus. We used YFP-IGFBP-3 fusion proteins lacking a signal prepeptide so that they remain intracellular and do not need to cross the plasma membrane to re-enter the cell. We confirmed that expressed YFP-IGFBP-3 fusion proteins were cell-associated and not present in the extracellular media. Confocal fluorescence microscopy showed that wild-type YFP-IGFBP-3 localized predominantly to the nucleus, whereas the NLS mutant YFP-MDGEA-IGFBP-3 was located predominantly in the cytoplasm. Expression of either the wild-type or mutant YFP-IGFBP-3 fusion protein induced PC-3 cell death as determined by loss of plasma membrane integrity. The loss of cell viability resulted from apoptosis since it was prevented by incubation with a broad-spectrum inhibitor of caspases, the proteases that are responsible for the characteristic changes of apoptosis. Similar results were obtained using IGFBP-3 mutants that do not bind IGFs (6m-IGFBP-3 and the double mutant, 6m/MDGEA-IGFBP-3). Together these results indicate that IGFBP-3 does not need to be secreted or concentrated in the nucleus to induce apoptosis in PC-3 cells in an IGF-independent manner.